In response to RFA-AI-11-004, this application proposes a highly collaborative research plan to use small molecule chemical inhibitors as probes to characterize the physiologic processes in Mycobacterium tuberculosis that are regulated by the essential Serine/Threonine protein kinases PknA and PknB. These kinases are both candidate drug targets themselves, and regulate downstream proteins and pathways that will also be potential targets for drug development. The PI and co-investigators will bring together state of the art expertise in transcriptomics, phosphoproteomics, lipidomics, metabolomics and computational biology with the goal of achieving a systems-level understanding of the PknA/PknB signaling network. In addition to this multi-platform omics approach, an important strength of the proposal is the use in this research of novel highly potent chemical inhibitors of these kinases as probes for PknA/PknB-regulated networks. The research proposes both unbiased, broad approaches to identify processes regulated by these kinases, and targeted approaches focused on two essential cell envelope biosynthetic pathways for which substantial data indicates a regulatory role for PknA and PknB: peptidoglycan synthesis and mycolic acid synthesis. While each research platform will generate data that will provide direct insight into the functions of PknA and PknB, another key component of this approach is to use computational methods to model networks that integrate the different types of experimental data. The results of this research will provide systems-level insights into the PknA/PknB signaling pathways and the processes that they regulate. We expect this knowledge will validate these kinases as excellent targets for M. tuberculosis drug development and identify proteins and processes regulated PknA and PknB that may also be attractive targets for anti-tuberculosis drug development. PUBLIC HEALTH RELEVANCE: Tuberculosis is a major cause of morbidity and mortality throughout the world, and drug-resistant tuberculosis is a major and increasing obstacle to successful treatment of infected individuals and to global efforts to control tuberculosis. By characterizing two M. tuberculosis regulatory proteins that are excellent candidates for drug development, this research will contribute to ongoing and future efforts to develop new drugs for the treatment of tuberculosis.